The present invention pertains in general to methods for purifying interferon produced by recombinant technology and to the products of the purification, and in particular to methods for purifying human immune interferon and a purified human immune interferon 4A.
The interferons form a family of antiviral and immunoregulatory proteins which are known to be naturally produced by fibroblasts, epithelial cells, and types of white blood cells called macrophages and lymphocytes. The three identified types of interferon are referred to as alpha (leukocyte), beta (fibro-epithelial), and gamma (immune).
All three types of interferon are being investigated for use in the treatment of human diseases. As a result, it is important to highly purify interferon in order to remove contaminants which might lead to side effects such as fever and allergic reactions. Despite the fact that all of the types of interferon presumably have common structural and chemical properties, no single method has been found to be effective in purifying all interferons.
Techniques for purifying interferons include affinity chromatography (controlled pore glass, CPG; zinc chelates; concanavalin A; anti-interferon antibodies; and acrylonitrile high polymers [Nobuhara, et al., U.S. Pat. No. 4,465,622]; and affinity chromatography plus gel filtration chromatography [Yip, et al., Proc.Natl.Acad.Sci. USA, 78: 1601-1605 (1981)]. These techniques have been used to obtain lymphocyte-produced human immune interferon having a specific activity estimated to be about 10.sup.7 units per milligram of protein. Yip, et al., supra. However, lymphocytes are difficult to culture and the amount of interferon economically producible from cultures of lymphocytes is far less than that producible by means of recombinant technology.
Synthetic and cDNA genes coding for human immune interferon (IFN-.gamma.) have been inserted into plasmid vectors, introduced into procaryotic and eucaryotic hosts, and used to produce recombinant human immune interferons (rIFN-.gamma.) in "mature" and analog forms. "Mature" rIFN-.gamma. has an amino acid sequence of 146 residues, the amino terminal of which begins Cys-Tyr-Cys, based upon determination of the sequence of the gene encoding human immune interferon. Goeddel, et al., European patent application No. 077670. Various analogs of mature rIFN-.gamma. are described in Alton, et al., published PCT patent application No. WO83/04053. Among those described are analogs lacking the amino terminal Cys-Tyr-Cys residues, i.e., [des-Cys.sup.1 -Tyr.sup.2 -Cys.sup.3 ] IFN-.gamma. analogs. See also, Alton, et al., in "The Biology of the Interferon System 1983", De Maeyer, et al., eds., pages 119-128, Elsevier Science Publishers (1983), referring to "IFN-.gamma.4" which is [des-Cys.sup.1 -Tyr.sup.2 -Cys.sup.3 -Lys.sup.81 ] IFN-.gamma. expressed in Met.sup.-1 form. As ordinarily directly expressed in E.coli , rIFN-.gamma. differs from the natural IFN-.gamma. produced by lymphocytes in having an N-terminal methionine residue and in not being glycosylated. Thus, mature rIFN-.gamma. is directly expressed as [Met.sup.-1 ] IFN-.gamma. and the recombinant-produced analog [des-Cys.sup.1 -Tyr.sup.2 -Cys.sup.3 ] IFN-.gamma. (hereinafter referred to as "recombinant human immune interferon 4A" or "IFN-.gamma.4A") is expressed as [Met.sup.-1, des-Cys.sup.1 -Tyr.sup.2 -Cys.sup.3 ] IFN-.gamma..
The native form of IFN-.gamma. is reported to be a 40,000 to 60,000 molecular weight oligomer, which is presumably a dimer of two reported monomeric forms having molecular weights of 20,000 and 25,000. Removal of the carbohydrate moieties of the monomers by glycosidase treatment produces monomers of 16,000 and 18,500 molecular weight, respectively. Le, et al., J.Immunol., 132: 1300-1304 (1984). A monomeric form of rIFN-.gamma. has been calculated to have a molecular weight of 17,140. The differences in molecular weight between natural and recombinant forms IFN-.gamma. may be explained, at least in part, by the fact that natural IFN-.gamma. has undergone processing which has removed C-terminal amino acid residues, while the rIFN-.gamma. contains these additional residues.
Any procedure for the isolation of IFN-.gamma. must take into account the potential instability of IFN-.gamma. upon acid treatment. For example, Yip, et al., supra, observe that natural IFN-.gamma. exhibits an almost ten-fold drop in antiviral activity upon dialysis against a pH 2 solution followed by a neutral phosphate buffer. This suggests that IFN-.gamma. is denatured in acid and does not refold into the native structure.
A further complication in the purification of rIFN-.gamma. is involved with its extraction from E.coli. While natural IFN-.gamma. may be harvested from the medium surrounding cultured lymphocytes, E.coli rIFN-.gamma. is harvested by breaking open bacterial cells with a consequent release of proteolytic enzymes which may degrade the interferon produced. Denaturants, such as urea and guanidine-HCl, inhibit enzyme activity without irreversible loss in the activity of interferon during extraction. Kung, U.S. Pat. No. 4,476,049.
However, once denatured (unfolded), the appropriate conditions for refolding interferon are not readily determined. Consequently, although one approach to renaturing soluble native protein such as immunoglobulin or methionine-prochymosin involves denaturation and dilution in an alkaline solution of urea or guanidine hydrochloride and renaturation by reducing the pH below a pH effective to denature the protein [see, e.g., Lowe, et al., U.K. patent application No. GB 2138004A], the application of such techniques to interferon is not straightforward.
Consequently, it is desirable to have a method for purifying interferon, particularly rIFN-.gamma., such that it is provided in highly active forms.